Interplay between Flavodiiron Proteins and Photorespirationin Synechocystis sp. PCC 6803*□S

Received for publication, January 20, 2011, and in revised form, May 17, 2011 Published, JBC Papers in Press, May 20, 2011, DOI 10.1074/jbc.M111.223289

Yagut Allahverdiyeva‡, Maria Ermakova‡, Marion Eisenhut‡1, Pengpeng Zhang‡, Pierre Richaud§¶�,Martin Hagemann**, Laurent Cournac§¶�, and Eva-Mari Aro‡2

From the ‡Laboratory of Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku,Finland, §Commissariat a l’Energie Atomique (CEA), Direction des Sciences du Vivant, Institut de Biologie Environementale etBiotechnologie, Laboratoire de Bioenergetique et Biotechnologie des Bacteries et Microalgues, CEA Cadarache, F-13108 Saint-Paul-lez-Durance, France, ¶CNRS, UMR Biologie Vegetale et Microbiologie Environnementales, F-13108 Saint-Paul-lez-Durance, France,�Aix-Marseille Universite, F-13108 Saint-Paul-lez-Durance, France, and the **Institut Biowissenschaften, Pflanzenphysiologie,Universitat Rostock, Albert-Einstein-strasse 3, D-18059 Rostock, Germany

Flavodiiron (Flv) proteins are involved in detoxification of O2and NO in anaerobic bacteria and archaea. Cyanobacterial Flvproteins, on the contrary, function in oxygenic environment andpossess an extra NAD(P)H:flavin oxidoreductase module. Syn-echocystis sp. PCC6803 has four genes (sll1521, sll0219, sll0550,and sll0217) encoding Flv proteins (Flv1, Flv2, Flv3, and Flv4).Previous in vitro studies with recombinant Flv3 protein fromSynechocystis provided evidence that it functions as aNAD(P)H:oxygen oxidoreductase, and subsequent in vivo studies withSynechocystis confirmed the role of Flv1 and Flv3 proteins in theMehler reaction (photoreduction of O2 to H2O). Interestingly,homologous proteins to Flv1 and Flv3 can be found also in greenalgae, mosses, and Selaginella.Here, we addressed the functionof Flv1 and Flv3 in Synechocystis using the �flv1, �flv3, and�flv1/�flv3mutants and applying inorganic carbon (Ci)-depri-vation conditions. We propose that only the Flv1/Flv3 het-erodimer form is functional in theMehler reaction in vivo. 18O2labeling was used to discriminate between O2 evolution in pho-tosynthetic water splitting and O2 consumption. In wild type,�20% of electrons originated from water was targeted to O2under air level CO2 conditions but increased up to 60% in severelimitation of Ci. Gas exchange experiments with �flv1, �flv3,and �flv1/�flv3 mutants demonstrated that a considerableamount of electrons in these mutants is directed to photorespi-ration under Ci deprivation. This assumption is in line withincreased transcript abundance of photorespiratory genes andaccumulation of photorespiratory intermediates in theWT andto a higher extent in mutant cells under Ci deprivation.

Flavodiiron proteins (or A-type flavoproteins, Flv)3 areinvolved in the detoxification of O2 or NO in strict and faculta-tive anaerobic prokaryotes (1). A similar type of Flv proteins,however, with strict selectivity toward O2, was recently identi-fied also in some eukaryotic pathogenic protozoa (2). All ofthese Flv proteins contain two redox centers: a flavin mononu-cleotide, functioning as an electron acceptor, and a non-hemeFe-Fe center as an active site (1). Available crystal structures ofthe Flv proteins from prokaryotic and eukaryotic organismscontain two monomers in a “head-to-tail” arrangement, bring-ing the flavin mononucleotide and Fe-Fe centers of opposingmonomers into close proximity (3, 4).The genome sequence of the cyanobacterium Synechocystis

has revealed four genes (sll1521, sll0219, sll0550, and sll0217)encoding Flv proteins (Flv1, Flv2, Flv3, and Flv4, respectively).Unlike the organismsmentioned above, cyanobacterial Flv pro-teins possess an additional NAD(P)H:flavin oxidoreductasemodule fused at the C terminus (1, 5). Two Flv proteins inSynechocystis, Flv2 and Flv4, are unique for cyanobacteria.Interestingly, homologous proteins to Flv1 and Flv3 can befound also in green algae, mosses, and Selaginella (6), whereasonly one protein with very low similarity to Flv proteins hasbeen found in plant genomes (7). We have recently demon-strated an essential role of Flv2 and Flv4 in photoprotection ofthe photosystem (PS) II complex under ambient CO2 condi-tions (6). Nevertheless, the exact mechanism underlying thisphotoprotective effect and the localization of the Flv2 and Flv4proteins still remain to be elucidated.In vitro studies with recombinant Flv protein from Syn-

echocystis provided evidence that Flv3 functions as aNAD(P)H:oxygen oxidoreductase (5), and subsequent in vivo studies withSynechocystis confirmed the role of Flv1 and Flv3 proteins inphotoreduction of O2 (7). Photoreduction of O2 to hydrogenperoxide (H2O2) at the reducing side of PSI complex was dis-covered 60 years ago by Mehler in spinach chloroplasts andtherefore is called a Mehler reaction (8). Subsequently, it hasbeen reported that the primary product of the Mehler reaction

* This work was supported by Academy of Finland Center of Excellence Pro-ject 118637, the European Union Project Solar-H2 (FP7 Contract 212508),CNRS/Japan Science and Technology Agency (Program “Structure andFunction of Biomolecules: Response Mechanism to Environments”), andthe Nordic Energy Research Program (Project Nordic BioH2). This work wasalso supported by the HelioBiotec platform (funded by the EuropeanUnion (European Regional Development Fund)), the Region ProvenceAlpes Cote d’Azur, the French Ministry of Research, and the Commissariat al’Energie Atomique.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S4.

1 Present address: Institute of Plant Biochemistry, Heinrich-Heine-Universitat,D-40225 Dusseldorf, Germany.

2 To whom correspondence should be addressed: Laboratory of MolecularPlant Biology, Dept. of Biochemistry and Food Chemistry, University ofTurku, FI-20014 Turku, Finland. Fax: 358-23338075; E-mail: evaaro@utu.fi.

3 The abbreviations used are: Flv, A-type flavodiiron proteins; Ci, inorganiccarbon; ROS, reactive oxygen species; RT-q-PCR, real-time quantitative RT-PCR; Chl, chlorophyll; IAC, iodoacetamide; LHCII, light-harvesting complex;PSI, photosystem I; PSII, photosystem II; 2PG, 2-phosphoglycolate; Rubisco,ribulose-1,5-bisphosphate carboxylase/oxygenase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 27, pp. 24007–24014, July 8, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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http://www.jbc.org/content/suppl/2011/05/20/M111.223289.DC1.html Supplemental Material can be found at:

is superoxide anion (O2�), and its disproportionation produces

H2O2 and O2 (9). It is important to note that reactive oxygenspecies (ROS) produced during photoreduction of O2 do notaccumulate in intact chloroplasts due to efficient ROS-scaveng-ing enzymes in near proximity to the photosynthetic electrontransport chain (10). Contrary to theMehler reaction in plants,the cyanobacterialMehler reaction driven by Flv1 andFlv3 doesnot produce ROS (7).In addition to theMehler reaction and dark respiration, pho-

torespiration consumes O2 in oxygenic photosynthetic organ-isms (11) but, in this case, also liberates CO2. Photorespirationis based on the dual function of Rubisco, the key enzyme ofphotosynthetic carbon assimilation, which binds either CO2 orO2 to the active site depending on the partial pressure of thesegases. Oxygenation of ribulose-1,5-bisphosphate by Rubiscoproduces toxic 2-phosphoglycolate (2PG), which is metabo-lized by the photorespiratory pathway. Photorespiration isoften described as one of themostwasteful reactions due to lossof photosynthetically fixed carbon and energy (12). Cyanobac-teria have evolved sophisticated CO2-concentrating mecha-nisms that operate to increase the CO2 concentration aroundRubisco, inside of the subcellular compartment called the car-boxysome (13–16). The presence of efficient CO2 concentrat-ingmechanisms suggested that the photorespiratory pathway isprobably not essential in cyanobacteria (17). Nevertheless,more recent studies on Synechocystis mutants defective in allthree predicted photorespiratory pathways, the plant-like C2cycle and the bacterial-type glycerate and decarboxylationpathways, demonstrated a highCO2-requiring phenotype com-parable with plant photorespiratory mutants and thus sup-ported the existence and essential function of photorespiratory2PG metabolism in cyanobacteria (18, 19). In addition to 2PGdetoxification, the photorespiratory 2PG metabolism togetherwith theMehler reaction supports the acclimation of cyanobac-teria to high light stress because a double mutant �flv3/�gcvTdefective in both the Flv3 protein and the glycine decarboxylasecomplex, which is involved in plant-like C2 photorespiratorypathway, could not segregate completely, and themutant dem-onstrated a high light-sensitive phenotype (20).However, despite the well defined routes for 2PG metabo-

lism, the experimental evidence for photorespiratory gasexchange in cyanobacteria is still missing. Here, for the firsttime to the knowledge of the authors, we report photorespira-tory oxygen uptake by Rubisco under Ci deprivation conditionsin Synechocystismutant strains deficient in the flv1 and/or flv3genes.

EXPERIMENTAL PROCEDURES

Strain and Culture Conditions—Synechocystis sp. PCC 6803(WT) and mutant strains were grown in BG-11 medium buff-ered with 20mMN-[Tris(hydroxymethyl)methyl]-2-aminoeth-anesulfonic acid-KOH, pH8.2, with gentle agitation under con-tinuous light (50 �mol photons m�2 s�1) at 30 °C and 3% CO2.Three days before the experiments, the cells were shifted to airlevel CO2 atmosphere. For measurements, the cells were har-vested and resuspended in a fresh BG-11 medium withoutNaHCO3 at chlorophyll (Chl) concentration of 10–20�gml�1.Only when indicated, NaHCO3 was added. For Ci deprivation

experiments, the cells were incubated under the light in an air-tight Eppendorf tube for 4 h.Mutants—The construction of the single mutants �flv1

(containing a chloramphenicol-resistant cassette) and �flv3(containing a spectinomycin-resistant cassette) was describedpreviously (7). The doublemutant of�flv1/�flv3was generatedby second transformation of the�flv3mutant with themutatedflv1 construct. The genotype of all mutants was verified by PCRusing total chromosomal DNA and gene-specific primers forflv1 and flv3 (20), respectively (supplemental Fig. S1).Oxygen Evolution—Steady-state oxygen evolution rates were

measured with a Clark type oxygen electrode (DW1, Hansat-ech) at a saturating light intensity. To measure the net photo-synthesis, the cells were suspended in BG-11 medium supple-mented with 10 mM NaHCO3, and for the PSII activitymeasurements, 2mM 2,5-dimethyl-p-benzoquinone was addedto cell suspension as an artificial electron acceptor.P700 Light Curve Measurements—P700 measurements were

performed by DUAL-PAM-100 (Walz, Germany). The yield ofnon-photochemical energy dissipation in the PSI centersoccurring due to the acceptor side limitation of PSI, Y(NA),were calculated as Y(NA) � (Pm � Pm�)/Pm. For determinationof Pm, a saturating pulse (6000 �mol photons m�2 s�1, 300ms)was applied on cell samples preilluminated with far-red light(75 wattsm�2, 10 s). Thus, Pmwas defined as amaximal changeof the P700 signal upon transformation of P700 from the fullyreduced to the fully oxidized state. Pm� represents the maximalchange of the P700 signal upon application of a saturatingpulses at different increasing light intensities duration of 30 s.Membrane Inlet Mass Spectrometry—Measurements of 16O2

(mass 32), 18O2 (mass 36) and CO2 (mass 44) exchange wereperformed by membrane inlet mass spectrometry directly onculture suspensions. For analyses, 1.5-ml samples were intro-duced into the measuring chamber that is connected to thevacuum line of a mass spectrometer (model Prima-B, ThermoFisher Scientific) by a Teflon membrane located at the bottomof the chamber. With this setup, gases dissolved in the liquidabove the membrane diffuse through the thin Teflon layer andare directly introduced into the ion source of the mass spec-trometer. The measuring chamber was thermostated (30 °C)using a water jacket, and the suspension was continuouslymixed by a magnetic stirrer. Actinic light (500 �mol photonsm�2 s�1) was applied by a LED-powered fiber optic illuminator(PerkinElmer Life Sciences) when needed. Calculation of light-induced oxygen uptake is based on the fact that by photosyn-thetic activity the cells produce essentially 16O2 from water,whereas if the heavy isotope 18O2 is present in the suspension,its consumption reflectsO2 uptake.Using amixture of isotopes,photosynthetic and respiratory activity of cells can then be cal-culated simultaneously upon illumination using simple isotoperatio expressed either as exchange rates (uptake or productionflow rates) or as cumulative O2 exchange (amounts that havebeen produced or consumed as a function of time) as detailed inRef. 21. In our experiments, 18O2 (99% 18O2 isotope content,Euriso-Top) was injected by bubbling at the top of the suspen-sion just before vessel closure and gas exchangemeasurements.Real-time Quantitative RT-PCR (RT-q-PCR)—Total RNA

was extracted by TRIsure (Bioline) treatment and purified with

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1 unit of DNase (Ambion TurboDNase kit) to remove genomicDNA. Purified RNA (1 �g) was used for cDNA synthesis.Reverse transcription was performed with random hexamers(Promega) and SuperScript III Reverse Transcriptase (Invitro-gen) according to the manufacturer’s protocol. SynthesizedcDNAwas diluted 5-fold and used as a template for RT-q-PCR.The RT-q-PCR was performed on a Bio-Rad IQ5 system

using iQ SYBR Green Supermix (Bio-Rad) with 40 cycles ofamplification. Relative changes in gene expression were calcu-lated as described previously (6) using the constitutivelyexpressed rnpB gene as a control. The primers used in this studyare summarized in supplemental Table S1.Quantification of Gly and Ser—Free amino acids were

extracted from fresh cyanobacterial cell pellets of 2 ml of cul-ture with 80% ethanol at 65 °C for 3 h. After centrifugation, thesupernatants were dried by lyophilization and redissolved in 8mM Na2HPO4, pH 6.8. The content of Gly and Ser were deter-mined by HPLC as described in Ref. 22.Protein Analysis—A soluble fraction of proteins was isolated

as described (6). Protein complexes were separated using6–13% gradient blue native PAGE followed by immunoblottingwith the Flv3 protein-specific antibody.

RESULTS

Phenotype and Photosynthetic Properties of Synechocystis�flv1, �flv3, and �flv1/�flv3 Mutant Cells—Comparativegrowth characteristics of the WT and the �flv1, �flv3, and�flv1/�flv3 mutant strains were tested in BG11 medium atambient air. There was no difference between the WT and thesingle �flv1 and �flv3 or the double mutant �flv1/�flv3 strainsin their growth characteristics under the conditions describedabove under “Experimental Procedures” (data not shown).To compare the electron transfer capacities of theWT,�flv1,

�flv3, and �flv1/�flv3 strains, the whole chain photosyntheticelectron transfer activity and the PSII activity were monitored.In line with our previous report (6), the �flv1 and �flv3 singlemutants demonstrated slightly higher PSII activity in the pres-ence of an artificial electron acceptor, but the net photosynthe-sis rate was 8% lower as compared with the WT (Table 1). The�flv1/�flv3 doublemutant demonstrated only 89% of the activ-ity from water to NaHCO3 measured for the WT (Table 1).Furthermore, the PSII activity of the �flv1/�flv3 mutant wasslightly lower, 95% of that in the WT (Table 1).Because Flv1 and Flv3 proteins function in accepting elec-

trons beyond PSI, we next measured the yield of non-photo-chemical energy dissipation in PSI reaction centers Y(NA)

under high CO2 conditions. Fig. 1 demonstrates a typical lightresponse curve for WT with a sharp increase in Y(NA) at verylow light intensities, indicating a high acceptor side limitationof PSI due to an incomplete activation of Calvin-Benson cycle,themain terminal electron acceptor. Activation of CO2 fixationwith time and increasing light intensity is reflected in a declinein Y(NA). The �flv1 (Fig. 1), �flv3 and �flv1/�flv3 (data notshown) mutants demonstrated significantly higher Y(NA)parameter with increasing actinic light intensities comparedwith the WT.The results described above to demonstrate the acceptor side

limitation of PSI were verified by using methyl viologen as anartificial acceptor of electrons. Addition of methyl viologen toSynechocystis cell suspension before Y(NA) measurementscompletely eliminated the acceptor side limitation of PSI in the�flv1mutant cells (Fig. 1).Respiration, Photoreduction of O2 and CO2 Uptake in Flv

Mutants—Y(NA) measurements demonstrated an importantrole for the Flv1 and Flv3 proteins in alleviation of the acceptorside electron pressure in PSI. This could be assigned to thefunction of the Flv1 and Flv3 proteins in donation of electronstomolecular oxygen in theMehler reaction. This processmightbe strongly regulated also by other electron transfer routes. Toverify this, the WT and the Flv mutants were subjected todetailed gas exchange measurements. The respiratory O2uptake was monitored during the dark period, and the photo-reduction of O2 as well as the CO2 uptake were measured dur-ing the dark-light transition by applying the membrane inletmass spectrometry gas exchange analysis system (21). All Flvmutants, the �flv1 (17.5 �mol O2 mg Chl�1 h�1) and �flv3(16.1 �mol O2 mg Chl�1 h�1) single mutants as well as the�flv1/�flv3 (13.7 �mol O2mg Chl�1 h�1) double mutant dem-onstrated increased dark respiration rate as compared with theWT (10.4 �mol O2mg Chl�1 h�1, Table 1). On the other hand,theWT cells and all of the Flv mutants, �flv1, �flv3, and �flv1/�flv3, showed very similar CO2 uptake after switching on theactinic light (Fig. 2).

TABLE 1Photosynthetic activities of Synechocystis WT and the mutant cellsThe activity of PSII and the net photosynthetic rate were measured as the steady-state oxygen evolution rate in the presence of 2 mM 2,5-dimethyl-p-benzoquinoneand 10 mM NaHCO3, respectively. Values are given as a mean � S.D. from threeindependent experiments. Dark respirationwasmonitored bymembrane inletmassspectrometry, and values are given as �mol O2 mg Chl�1 h�1.

PSII activityNet photosynthetic

activity Dark respiration

% %WT 100 � 2.4 100 � 1.7 10.4 � 0.6�flv1 110 � 3.2 92 � 2.1 17.5 � 1.2�flv3 105 � 1.8 92 � 1.5 16.1 � 1.2�flv1/�flv3 95 � 2.4 89 � 1.2 13.7 � 0.7

FIGURE 1. Light response of the acceptor side limitation of PSI, Y(NA). Theacceptor side limitation of PSI was recorded from the WT (f) and �flv1 (F)mutant strain. 0.1 mM methyl viologen (E) was added to �flv1 cell suspen-sion before the measurement. Values are the mean � S.D. from threeindependent experiments.

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18O2 labeling of the cells was then used to separately analyzethe evolution and uptake of O2 during illumination of the cells.In theWT cells, illumination strongly stimulatedO2 uptake (28�mol O2mgChl�1 h�1) in the presence of 5mMNaHCO3 (Fig.3). Under the same conditions, the �flv1 and �flv3 singlemutant cells did not show any photoreduction ofO2, which is inline with previous studies (Fig. 3) (7). Likewise, the doublemutant �flv1/�flv3 was not able to consume O2 during illumi-nation in the presence of NaHCO3.The above experiments confirmed the involvement of both

the Flv1 and Flv3 proteins in theMehler reaction of Synechocys-tis. The next question to be addressed was whether there is acompetition between electrons from water oxidation to theMehler reaction, photorespiration, and CO2 fixation. To thisend, the cell culture was depleted of CO2, the main terminalelectron acceptor of the photosynthetic electron transfer chain.For this purpose, the cells were kept in tightly closed Eppendorftubes for 4 h. In the absence of dissolved CO2 and without

exogenously added bicarbonate (Ci deprivation), the WT cellsdemonstrated enhanced light-induced O2 uptake comparedwith that measured in control conditions in the presence ofbicarbonate (Fig. 3). Surprisingly, such severe Ci deprivationconditions were found to stimulate the photoreduction of O2also in the�flv1 and�flv3 single mutants. These data were firstunderstood to indicate that the Flv1 or Flv3 protein can func-tion in theMehler reaction as a homodimer under specific con-ditions such as Ci deprivation. To test this assumption, we gen-erated the double mutant �flv1/�flv3. Intriguingly, the �flv1/�flv3 double mutant also demonstrated a similar O2photoreduction rate under Ci deprivation conditions as the sin-gle mutants (Fig. 3), thus excluding the possibility of functionalFlv1 or Flv3 homodimers. The similar behavior of both the Flvmutants as well as the WT provided evidence that the O2 pho-toreduction under Ci deprivation conditions could have anorigin different from the Flv1- and Flv3-mediated Mehlerreaction.Because the enhancement of O2 photoreduction upon Ci

deprivation can, in principle, originate from enhanced functionof respiratory terminal oxidases, photorespiration, or electronflux from PSI or stromal components directly to O2 (classicalplant-type Mehler reaction), the origin of the O2 photoreduc-tion was further studied by applying specific inhibitors. Theaddition of KCN, an inhibitor of terminal oxidases, completelyabolished dark respiration (Fig. 4A) but did not significantlyaffect the photoreduction of O2 in the �flv1/�flv3mutant cells(Fig. 4B).Photoreduction of O2 was next measured in the presence of

iodoacetamide (IAC), a widely used inhibitor of CO2 fixationand photorespiration (7, 23). Measurements with cell suspen-sions supplemented with IAC were first performed in the pres-ence of bicarbonate. In such conditions, the addition of IACstrongly stimulated the rate of O2 photoreduction in WT (Fig.5A), whereas in the �flv1 (supplemental Fig. S2A) and �flv3(supplemental Fig. S2B) single mutants as well as in the �flv1/�flv3 double mutant (Fig. 5B) the stimulation of O2 photore-duction was only modest.We next proceeded to compare the effect of IAC on photo-

reduction of O2 at Ci deprivation conditions. As demonstratedpreviously in Fig. 3, the WT as well as all the Flv mutant cellsshowed strong O2 photoreduction at Ci deprivation (Fig. 5, C

FIGURE 2. Mass spectrometric measurements of CO2 uptake during dark-light transition. During measurement, the cells were first kept in darkness for5 min, and thereafter the white light of 500 �mol photons m�2 s�1 wasturned on (upward arrow) and off again (downward arrow).

FIGURE 3. Mass spectrometric measurements of O2 consumption duringdark-light transition in the WT, �flv1, �flv3, and �flv1/�flv3 mutants ofSynechocystis. The cells were kept in darkness for 5 min and then illuminatedwith white light of 500 �mol photons m�2 s�1 for 5 min to measure theuptake of O2. The asterisk represents cells demonstrated only trace amount of18O2 consumption. White bars, 5 mM NaHCO3 was present in the medium.Black bars, measurements were performed with the cells exposed to Ci dep-rivation. Values are the mean � S.D. from three independent experiments.

FIGURE 4. Effect of KCN on dark respiration and photoreduction of O2.Dark respiration (A) and O2 photoreduction (B) were monitored after additionof KCN (final concentration, 1 mM) (dashed line in B) to Ci-deprived �flv1/�flv3cells. Arrays indicate the addition of inhibitor (A) and switching on the actiniclight (B). In B, the cumulative O2 uptake curve is calculated from 18O2/16O2exchange measurements and is presented here after subtraction of the darkO2 uptake rate for better legibility.

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and D, for WT and �flv1/�flv3, respectively). It is conceivablethat theCi deprivation favors the oxygenation of RuBP and thusphotorespiratory 2PG metabolism. The addition of IAC to theCi-deprivedWT cell suspension to inhibit the CO2 fixation andphotorespiration did not significantly change O2 photoreduc-tion (Fig. 5C). In sharp contrast to the WT, addition of IAC tothe �flv1, �flv3 (supplemental Fig. S2, C and D, respectively),and �flv1/�flv3 double mutant cells abolished �75% of O2photoreduction (Fig. 5D). High sensitivity of O2 photoreduc-tion to IAC, the Calvin-Benson cycle inhibitor, during Ci dep-rivation, led us to conclude that the Ci-sensitive O2 photore-duction in the �flv1 and �flv3 mutants as well as in the �flv1/�flv3 double mutant is mainly due to photorespiration, i.e.oxygenase function of Rubisco. Similar results were obtainedwhen cell suspension was supplemented with glycolaldehyde(supplemental Fig. S4), another effective inhibitor of the pho-tosynthetic carbon reduction cycle (24). About 25% of O2 pho-toreduction in the �flv1, �flv3, and �flv1/�flv3 mutant cells

was insensitive to IAC (Fig. 5D), and this might originate fromthe electron flux from the PSI complex directly to O2 or from afraction of Rubisco that was not fully inhibited by the inhibitor.It was also tested that IAC has no effect on the function ofterminal oxidases in the dark (Fig. S3).Photorespiratory Pathway—To support the assumption of an

activated photorespiratory 2PG cycle in Ci-limitedWT and Flvmutant cells, the relative expression level of two genes, gcvT(sll0171) and ilvB (sll1981), encoding the Gly decarboxylasecomplex T protein and glyoxylate carboligase, involved in theplant-like C2 cycle and bacterial-type glycerate photorespira-tory pathways, respectively, were analyzed by RT-q-PCR. Therelative transcript abundance of these genes was studied inthe WT and �flv1/�flv3 mutant cells from three differentconditions: (i) high CO2 grown cells where the occurrence ofphotorespiratory pathway is minimal, (ii) after Ci depriva-tion treatment where enhanced photorespiratory metabolismis expected, and (iii) after addition of IAC to Ci-deprived cells.Under Ci deprivation conditions, the transcript levels of thegcvT and ilvB genes were�2–3-fold higher in both theWT and�flv1/�flv3 mutant cells as compared with high CO2 growncells (Fig. 6A). On the contrary, addition of IAC significantlydecreased the transcript level of both genes. It is important tonote that even in high CO2 grown cells, the transcripts of gcvTand especially of ilvB genes were much more abundant in the�flv1/�flv3mutant cells as compared with the WT.Gly and Ser, the photorespiratory intermediates, were also

monitored in theWTandmutant cells. After 12 h of incubationunder Ci deprivation conditions, all cells showed a clear accu-mulation of Gly and Ser, indicating an enhanced flux into theplant-like C2 photorespiratory cycle. AlthoughWT cells accli-mate after longer times, resulting in a decrease in Gly content,the�flv1 and�flv3mutant cells still demonstrated a high accu-mulation of both amino acids (Fig. 6B). Obviously, severe Cideprivation does lead to the accumulation of intermediates ofthe plant-like 2PG pathway despite the slight increase in tran-script level of some photorespiratory genes during the activephotorespiration.Dimerization of Flv Proteins—Because the Flv proteins from

different organisms have demonstrated a homodimer or homo-tetramer organization in vitro (3–5), we addressed the questionabout dimerization of Synechocystis Flv proteins. Both Flv1 and

FIGURE 5. Effect of IAC on photoreduction of O2 in the WT and �flv1/�flv3cells. O2 photoreduction was monitored in the presence of 5 mM NaHCO3(solid line) and in the presence of both 5 mM NaHCO3 and 8 mM IAC (dashedline) from the WT (A) and �flv1/�flv3 mutant cells (B). Similar measurementswere performed from the WT (C) and �flv1/�flv3 (D) cells after Ci deprivation.Arrays indicate switching on the light. Cumulative O2 uptake curve was cal-culated from 18O2/16O2 exchange measurements and is presented here aftersubtraction of dark O2 uptake rate for better legibility.

FIGURE 6. Effect of Ci deprivation on photorespiratory markers. A, transcript abundance of the gcvT and ilvB genes in the WT (black bars) and �flv1/�flv3(white bars) cells. Samples were taken from the cells grown in the presence of 3% CO2 (HC), after 12 h shift from HC to Ci deprivation conditions (Ci-dep) and after4 h treatment with IAC of the samples incubated at Ci deprivation for 8 h (�IAC). r.u., relative units. B, accumulation of Gly and Ser in the WT, �flv1, and �flv3cells. Samples were taken from the cells grown in the presence of 3% CO2, after a 12- and 36-h shift from HC to Ci deprivation conditions.

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Flv3 are soluble proteins in the cytoplasm (6). To determinewhether the Flv1 and Flv3 proteins are dimerized in vivo, thesoluble protein fraction was isolated from the WT and �flv1mutant cells, subjected to blue native PAGE, and immuno-blotted with the Flv3 antibody. As shown in Fig. 7B, the Flv3antibody in the WT cells recognized a major band above the140-kDa marker, suggesting that most of the Flv3 proteins arepresent as a homo- or heterodimer. However, the Flv1 antibodydid not recognize the protein in Synechocystis cell extracts butonly when overexpressed in Synechocystis or Escherichia coli,thus being in accordance with a very low transcript level of theflv1 gene in the cells (6). Accordingly, this antibody could not beused to analyze the 140-kDa band. The presence of a proteinband with similar molecular mass also in the �flv1mutant cellsindicated that the Flv3 protein is capable of forminghomodimers as well. However, the similar behavior of the�flv1/�flv3 doublemutant and both of the Flv singlemutants inthe photoreduction of O2 in the presence of bicarbonate and inCi deprivation, suggested that the Flv3 or Flv1 homodimers arenot active in theMehler reaction in vivo. In line with functionalsignificance of the heterodimer, the accumulation of the Flv3protein was dependent on the presence of the Flv1 protein, asthe amount of the Flv3 protein was significantly down-regu-lated in the �flv1mutant cells (Fig 7B).

DISCUSSION

Mehler Reaction Is a Strong Sink of Electrons in Cyano-bacteria—In plant chloroplasts, the Mehler reaction plays aprotective role acting as a sink for excess electrons under severestress conditions, for example when CO2 fixation is impaired

(25). This reaction leads to the production of ROS. As a conse-quence, plants have during the evolution developed a strongROS-scavenging system (26). It is intriguing to note that cyano-bacteria, the progenitors of chloroplasts, employ a completelydifferent strategy for performing Mehler reactions. The Flv1and Flv3 proteins, performing theMehler reaction in cyanobac-teria (Fig. 3), can reduce O2 to water without generation anyROS. It has been proposed that the Mehler reaction in cyano-bacteria is evolutionarily related to the response of anaerobicbacteria to O2 (7).Assuming that the O2 photoreduction in the WT Syn-

echocystis cells in the presence of saturated amount of Ci ismainly due to the Mehler reaction operated via Flv1 and Flv3,the cells pass �20% of electrons originating from PSI to O2 viathe Flv1/Flv3 heterodimer. At Ci deprivation conditions, theO2photoreduction rate was 50–60% of gross photosynthetic O2evolution in the WT Synechocystis cells. Because IAC, theinhibitor of CO2 fixation and photorespiration, was found tohave a stimulatory effect on photoreduction of O2, to a largerextent in the presence of Ci than in Ci deprivation, the capacityof the Mehler reaction is likely to be very high. This is deducedfrom the fact that IAC, by inhibiting the CO2 uptake in thepresence of Ci and suppressing possible electron flux via pho-torespiration under the Ci deprivation, stimulatesMehler reac-tion to a larger extent in the WT. Thus, the Mehler reactionseems to function as an efficient sink of electrons and therebydissipates excess energy in the photosynthetic apparatus of Syn-echocystis. Nevertheless, it is likely that cyanobacteria with effi-cient CO2 concentrating mechanism only seldom use theirMehler reaction in its full capacity.Even though dimerization is a general feature of Synechocys-

tis Flv proteins, only the Flv1/Flv3 heterodimer was found to bea physiologically functional form in theMehler reaction in vivo.A significant decrease in the amount of Flv3 in the�flv1mutantalso suggests that the stability of the Flv1 and Flv3 proteins isco-regulated. We do not, however, exclude the possibility thathomodimers of Flv1 and Flv3 might be formed at least in themutant background that could have another, still unknownfunction in cells.Evidence for Functional Photorespiratory Gas Exchange in

Cyanobacteria—In addition to theMehler reaction inWTcells,it was intriguing to note that the Flvmutants are also capable todirect 40–60% of electrons from the photosynthetic electrontransfer chain to photoreduction of O2. At first glance, theseresults are striking and even contradictory. It should be noted,however, that such strong photoreduction of O2 was recordedonly at severe limitation of Ci under conditions where the WTefficiently directs electrons mainly to the Mehler reaction.Indeed, there is compelling evidence that the mutant strainswith inactivated flv1 and/or flv3 gene do not perform light-induced O2 uptake when Ci is present as a terminal electronacceptor (Fig. 3) (7). Therefore, significant O2 photoreductionunder severe Ci deprivation in both the �flv1 and �flv3mutantcells as well as in the double mutant must have a differentorigin.In all cyanobacteria, two partially overlapping photorespira-

tory pathways, one similar to plant photorespiratory metabo-lism and the other similar to bacterial glycerate pathway, have

FIGURE 7. Dimerization of the Flv1 and Flv3 proteins in the WT andmutant cells. Soluble fraction of cellular proteins was isolated as described(6). Protein complexes were separated using 6 –13% gradient blue nativePAGE electrophoresis (A) and immunodetected with Flv3 antibody (B). Thearrow indicates the location of the Flv3 protein where it is present as a dimerwith molecular mass of �140 kDa. The molecular mass of the Flv monomer is70 kDa. Gels were loaded on a protein basis.

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been predicted from in silico analyses (19). A third metabolicroute, the decarboxylation pathway, was suggested recently byEisenhut et al. (18). It was hypothesized that the photorespira-tory 2PGmetabolism is important for distribution of the crucialintermediate glyoxylate for the synthesis of metabolic precur-sors and biomass. Recent computational simulations of theSynechocystis primary metabolism demonstrated that themajority of Gly and Ser originate from photorespiratory 2PGmetabolism at photoautotrophic growth. Only under dark con-ditions, themuch lower glyoxylate demandmay be alternativelyfulfilled by proline catabolism (27).Cyanobacteria are evolutionarily the first prokaryotes per-

forming water oxidation, which resulted in high intracellularO2 level. A strong focus during evolution of oxygenic photosyn-thetic organisms has been on preventing the damages of self-generated oxidative environment. Additionally, in nature cya-nobacteria are often exposed to Ci limitation (28) conditions(for instance, acidic water, low mixing, cyanobacterial mats,etc.), and this would favor the cells to perform photorespirationat higher rates. It is conceivable that the efficient function of theFlv1 and Flv3 proteins in the Mehler reaction in cyanobacteriareduces but does not eliminate the importance of photorespi-ration in dissipation of excess energy under low Ci conditions.The application of 18O2 labeling and the Ci deprivation condi-tions to the mutant cells without Mehler background clearlydemonstrated that the Flv-based Mehler reaction is not theonly sink for electrons in Ci deprivation. Indeed, a considerableamount of electrons under such conditions are directed to pho-torespiratory 2PG metabolism in the �flv1, �flv3, and �flv1/�flv3mutants. Moreover, our results do not exclude but ratheralso support a possibility that in WT Synechocystis cells, a cer-tain amount of electrons is directed to the detoxification of theoxygenation product 2PG of Rubisco under specific conditions,such as Ci deprivation. In line with our present results, an inter-action of the Flv3 protein with photorespiration was suggestedto be crucial based on the high light-sensitive phenotype of thedouble mutant �flv3/�gcvT (20).Evolutionary Implications on Elimination of Cyanobacterial-

type and Development of Plant-type Mehler Reaction—Genesencoding the Flv1 and Flv3 type proteins have been found insequenced genomes of all cyanobacteria aswell as in green algaeandmosses (6). During evolution of higher plants, the Flv-basedMehler reaction, with water as a direct end product, was lostand became replacedwith the plant-type, classicalMehler reac-tion, which produces ROS, and evolved in concert with an effi-cient ROS-scavenging system. The plant-type Mehler isunlikely to support significant electron flow, with the estimatedvalues being less than 10% (11), and its function is clearly lim-ited as an electron sink under severe stress conditions. Ineukaryotic photosynthetic organisms, the excess energy dissi-pation and adjustment of the NADPH/ATP ratio is in closecommunication withmitochondria, whichmight in some casesdivert excess reducing power into (distant) O2 consumption(29). It is also likely that the elimination of Flv proteins in higherplant chloroplasts was closely related to the movement of lifefromwater to the land. Life in the atmosphere affords intensivegas diffusion, thus avoiding overaccumulation of O2 in photo-synthetic cells and probably making the Flv proteins dispensa-

ble. It is conceivable that in higher plants, the regulation of theredox poise in chloroplasts by cyclic electron flow around PSItogether with efficient non-photochemical quenching andlight-harvesting complex II phosphorylation is enough to pro-vide sufficient photoprotection (30). In aquatic or humid envi-ronments, gas diffusion is slower; therefore, a direct scavengingof O2 via the Flv proteins and without formation of ROS isprobablymore important for efficient carboxylation of Rubisco.Green algae and mosses represent evolutionary intermediates.Their dissipation of excess energy, for instance by the non-photochemical quenching system, resembles that of higherplants (31–33), but they still harbor the genes involved in theFlv-type Mehler reaction (6). Photorespiratory pathway alsofunctions as an efficient electron sink for dissipation of excessenergy inC3plants, particularly under limitingCO2 conditions,and accordingly plays a crucial role also in high light acclima-tion of C3 plants (34, 35). In cyanobacteria, on the contrary, theconcerted function of the Flv proteins has themost decisive rolein dissipation of excess energy in Ci deprivation and apparentlyalso during dark-light transitions.

Acknowledgments—We thank Dr. N. Battchikova for fruitful discus-sions and K.Michl for amino acid analyses. Professors A. Kaplan andT. Ogawa are thanked for kindly providing us with the Flv1 and Flv3knock-out mutants.

REFERENCES1. Vicente, J. B., Justino, M. C., Goncalves, V. L., Saraiva, L. M., and Teixeira,

M. (2008)Methods Enzymol. 437, 21–452. Vicente, J. B., Testa, F., Mastronicola, D., Forte, E., Sarti, P., Teixeira, M.,

and Giuffre, A. (2009) Arch. Biochem. Biophys. 488, 9–133. Di Matteo, A., Scandurra, F. M., Testa, F., Forte, E., Sarti, P., Brunori, M.,

and Giuffre, A. (2008) J. Biol. Chem. 283, 4061–40684. Frazao, C., Silva, G., Gomes, C. M., Matias, P., Coelho, R., Sieker, L.,

Macedo, S., Liu, M. Y., Oliveira, S., Teixeira, M., Xavier, A. V., Rodrigues-Pousada, C., Carrondo, M. A., and Le Gall, J. (2000) Nat. Struct. Biol. 7,1041–1045

5. Vicente, J. B., Gomes, C. M., Wasserfallen, A., and Teixeira, M. (2002)Biochem. Biophys. Res. Commun. 294, 82–87

6. Zhang, P., Allahverdiyeva, Y., Eisenhut, M., and Aro, E. M. (2009) PLoSOne 4, e5331

7. Helman, Y., Tchernov, D., Reinhold, L., Shibata, M., Ogawa, T., Schwarz,R., Ohad, I., and Kaplan, A. (2003) Curr. Biol. 13, 230–235

8. Mehler, A. H. (1951) Arch. Biochem. Biophys. 33, 65–779. Asada, K., Kiso, K., and Yoshikawa, K. (1974) J. Biol. Chem. 249,

2175–218110. Asada, K. (2006) Plant Physiol. 141, 391–39611. Badger, M. R., von Caemmerer, S., Ruuska, S., and Nakano, H. (2000)

Philos Trans. R. Soc. Lond B. Biol. Sci. 355, 1433–144612. Artus, N. N., Somerville, S. C., Somerville, C. R., and Lorimer, G. H. (1986)

CRC Crit. Rev. Plant Sci. 4, 121–14713. Badger, M. R., and Price, G. D. (2003) J. Exp. Bot. 54, 609–62214. Kaplan, A., and Reinhold, L. (1999) Annu. Rev. Plant Physiol. Plant Mol.

Biol. 50, 539–57015. Price, G. D., Badger, M. R., Woodger, F. J., and Long, B. M. (2008) J. Exp.

Bot. 59, 1441–146116. Battchikova, N., Eisenhut, M., and Aro, E. M. (2010) Biochim. Biophys.

Acta, in press17. Colman, B. (1989) Aquat Bot. 34, 211–23118. Eisenhut, M., Ruth, W., Haimovich, M., Bauwe, H., Kaplan, A., and Hage-

mann, M. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 17199–1720419. Bauwe, H., Hagemann, M., and Fernie, A. R. (2010) Trends Plant Sci. 15,

330–336

Photorespiration in Cyanobacteria

JULY 8, 2011 • VOLUME 286 • NUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 24013

at FIN

ELIB

- Turku U

niv, on July 20, 2011w

ww

.jbc.orgD

ownloaded from

20. Hackenberg, C., Engelhardt, A., Matthijs, H. C., Wittink, F., Bauwe, H.,Kaplan, A., and Hagemann, M. (2009) Planta 230, 625–637

21. Dimon, B., Gans, P., and Peltier, G. (1988) Methods Enzymol. 167,686–691

22. Hagemann, M., Vinnemeier, J., Oberpichler, I., Boldt, R., and Bauwe, H.(2005) Plant Biol. 7, 15–22

23. Wildner, G. F., and Henkel, J. (1976) Biochem. Biophys. Res. Commun. 69,268–275

24. Miller, A. G., and Canvin, D. T. (1989) Plant Physiol. 91, 1044–104925. Johnson, X., Wostrikoff, K., Finazzi, G., Kuras, R., Schwarz, C., Bujaldon,

S., Nickelsen, J., Stern, D. B., Wollman, F. A., and Vallon, O. (2010) PlantCell 22, 234–248

26. Foyer, C. H., and Noctor, G. (2000) New Phytol. 146, 359–38827. Knoop, H., Zilliges, Y., Lockau, W., and Steuer, R. (2010) Plant Physiol.

154, 410–422

28. Talling, J. F. (1985) in Inorganic Carbon Uptake by Aquatic PhotosyntheticOrganisms (Berry, W. J., ed) American Society of Plant Physiologists, pp.403–420, Rockville, MD

29. Noguchi, K., and Yoshida, K. (2008)Mitochondrion 8, 87–9930. Tikkanen, M., Grieco, M., and Aro, E. M. (2011) Trends Plant Sci. 16,

126–13131. Bonente, G., Passarini, F., Cazzaniga, S., Mancone, C., Buia, M. C.,

Tripodi, M., Bassi, R., and Caffarri, S. (2008) Photochem. Photobiol. 84,1359–1370

32. Alboresi, A., Gerotto, C., Giacometti, G.M., Bassi, R., andMorosinotto, T.(2010) Proc. Natl. Acad. Sci. U.S.A. 107, 11128–11133

33. Peers, G., Truong, T. B., Ostendorf, E., Busch, A., Elrad, D., Grossman,A. R., Hippler, M., and Niyogi, K. K. (2009) Nature 462, 518–521

34. Osmond, C. B. (1981) Biochim. Biophys. Acta 639, 77–9835. Kozaki, A., and Takeba, G. (1996) Nature 384, 557–560

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